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1. Introduction to Biochemistry4h 34m
2. Water3h 23m
3. Amino Acids8h 10m
4. Protein Structure10h 4m
5. Protein Techniques14h 5m
6. Enzymes and Enzyme Kinetics13h 38m
7. Enzyme Inhibition and Regulation 8h 42m
8. Protein Function 9h 41m
9. Carbohydrates7h 49m
10. Lipids5h 49m
11. Biological Membranes and Transport 6h 37m
12. Biosignaling9h 45m
Review 1: Nucleic Acids, Lipids, & Membranes2h 47m
Review 2: Biosignaling, Glycolysis, Gluconeogenesis, & PP-Pathway3h 12m
Review 3: Pyruvate & Fatty Acid Oxidation, Citric Acid Cycle, & Glycogen Metabolism2h 26m
Review 4: Amino Acid Oxidation, Oxidative Phosphorylation, & Photophosphorylation1h 48m

5. Protein Techniques

SDS-PAGE Strategies

5. Protein Techniques

SDS-PAGE Strategies: Videos & Practice Problems

Video Lessons Practice Worksheet

Topic summary

SDS PAGE is a technique that separates proteins based on mass, allowing for the analysis of protein subunits. Unlike native PAGE, SDS PAGE can separate non-covalently linked subunits, while covalently linked subunits require reduction with agents like beta mercaptoethanol to break disulfide bonds. In SDS PAGE, proteins are denatured by sodium dodecyl sulfate (SDS), which imparts a negative charge, enabling size-based separation. Understanding these interactions is crucial for studying protein structure and function, particularly in quaternary structures where subunits may be linked by both covalent and non-covalent interactions.

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SDS-PAGE Strategies

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SDS-PAGE Strategies Video Summary

SDS PAGE, or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is a powerful technique used to separate proteins based on their mass. Unlike native PAGE, which maintains the proteins' native structures and interactions, SDS PAGE denatures proteins, allowing for the analysis of individual subunits. This method is particularly useful for separating protein subunits that are not covalently linked. However, when covalent bonds, such as disulfide bonds, are present, additional steps are necessary to fully separate these subunits.

Beta mercaptoethanol (often abbreviated as βME) is a reducing agent that cleaves disulfide bonds, making it essential for analyzing proteins with quaternary structures. For instance, consider a protein composed of four subunits: A, B, C, and D. In this scenario, subunits A and C are linked by a disulfide bond, while subunits B and D are connected only through non-covalent interactions. When subjected to native PAGE, the entire protein complex appears as a single band due to the retention of its native properties, influenced by the shape, mass, and charge of the protein.

In contrast, when SDS is introduced, it denatures the proteins and imparts a negative charge, allowing them to migrate through the gel based solely on size. However, SDS does not break disulfide bonds, meaning that subunits A and C remain associated and travel together as a single band. This band will be the largest due to the combined mass of subunits A and C, while subunits B and D will separate based on their individual sizes, resulting in distinct bands for each.

To fully separate all subunits, βME is added prior to SDS PAGE. This treatment cleaves the disulfide bond between subunits A and C, allowing them to migrate independently. Consequently, the largest band will now represent only subunit A, followed by subunit B, and then subunit C, with subunit D remaining the smallest and fastest migrating band. This strategic use of βME in conjunction with SDS PAGE provides valuable insights into protein structure and interactions, enabling a clearer understanding of the individual components of complex proteins.

Problem

Compare the Native & SDS PAGE gels to indicate if each sample is a monomer, dimer, trimer or tetramer.

a. Sample 1:
b. Sample 2:
c. Sample 3:
d. Sample 4:

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Problem

'Protein X' has a molecular mass of 400 kDa when measured by size-exclusion chromatography. When subjected to SDS-PAGE, Protein X gives 3 bands with molecular masses of 180, 160, & 60 kDa. When SDS-PAGE is conducted a second time but in the presence of β-mercaptoethanol (β-ME), 3 bands form again, but this time with molecular masses of 160, 90, and 60 kDa. What is the subunit composition of Protein X? (Hint: draw both SDS-PAGE gels).

Protein X has 3 subunits with masses of 160, 90 & 60 kDa.

Protein X has 4 subunits with masses of 160, 90, 90 & 60 kDa.

Protein X has 3 subunits with masses of 180, 160, & 60 kDa.

Protein X has 4 subunits with masses of 180, 160, 90 & 60 kDa.

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What is the purpose of using SDS in SDS-PAGE?

SDS, or sodium dodecyl sulfate, is used in SDS-PAGE to denature proteins and impart a uniform negative charge. This ensures that proteins are separated based solely on their mass, rather than their shape or charge. SDS disrupts non-covalent bonds, causing proteins to unfold into linear chains. This uniform negative charge allows the proteins to migrate towards the positive electrode during electrophoresis, with smaller proteins moving faster through the gel matrix. This separation by size is crucial for analyzing protein subunits and understanding protein structure and function.

How does beta mercaptoethanol aid in SDS-PAGE analysis?

Beta mercaptoethanol is a reducing agent used in SDS-PAGE to break disulfide bonds between protein subunits. Disulfide bonds are covalent links that can hold protein subunits together. By cleaving these bonds, beta mercaptoethanol allows the individual subunits to separate and migrate independently through the gel. This is particularly useful for analyzing proteins with quaternary structures, where subunits may be linked by disulfide bonds. The separation of these subunits provides detailed information about the protein's composition and structure.

What is the difference between SDS-PAGE and native PAGE?

SDS-PAGE and native PAGE are both electrophoresis techniques used to separate proteins, but they differ in their approach. SDS-PAGE uses sodium dodecyl sulfate (SDS) to denature proteins and impart a uniform negative charge, allowing separation based solely on mass. In contrast, native PAGE maintains the proteins' native state, preserving their shape, charge, and mass. This means that in native PAGE, proteins are separated based on a combination of these factors. SDS-PAGE is useful for analyzing protein subunits, while native PAGE is better for studying protein complexes and interactions in their native form.

Why is it important to understand the migration of proteins in SDS-PAGE?

Understanding the migration of proteins in SDS-PAGE is crucial for accurately interpreting the results of the electrophoresis. Proteins are separated based on their size, with smaller proteins migrating faster through the gel. By analyzing the position of protein bands, researchers can determine the molecular weight of the proteins and identify different subunits. This information is essential for studying protein structure, function, and interactions. Additionally, knowing how proteins migrate helps in troubleshooting experimental issues and optimizing the conditions for better resolution and accuracy.

How can SDS-PAGE be used to study protein quaternary structure?

SDS-PAGE can be used to study protein quaternary structure by analyzing the individual subunits of a protein complex. In the presence of SDS, proteins are denatured and separated based on their size. If the protein subunits are linked by disulfide bonds, a reducing agent like beta mercaptoethanol can be used to break these bonds, allowing the subunits to separate and migrate independently. By comparing the migration patterns with and without the reducing agent, researchers can determine the composition and arrangement of the subunits, providing insights into the quaternary structure of the protein.